The overall objective of this proposal is to use protein crystallography to understand the structural basis for the catalytic efficiency of enzymes and how protein dynamics is coupled to protein function and thermostability. Enzymatic catalysis is central to all biochemical processes, and its defects are at the core of most inherited metabolic diseases. Detailed knowledge of how enzymes work should lead to better understanding of the biochemistry that underlies metabolic diseases and their treatment. Within this overall research program there are eight sub-projects. The specific aim of the first sub-project is to understand the protein structural features responsible for the catalytic efficiency of triosephosphate isomerase. Crystal structures of the enzyme from two sources complexed with substrates and inhibitors will be compared with structures of mutant enzymes generated by site-directed mutagenesis. Triosephosphate isomerase is a central enzyme in the metabolism of carbohydrates, and its deficiency leads to a multisystem disorder involving neurological disfunction, hemolytic anemia, and a propensity for sudden cardiac death. The second project aims to use a combination of crystallography and genetic selection to understand the relationship between protein dynamics and catalysis and thermostability. The target enzyme, 2-isopropyl malate dehydrogenase, is essential for the biosynthesis of leucine. Project 3 aims to develop methods for low-temperature crystallography of enzyme-substrate complexes, and also to map the dynamic properties of proteins as a function of temperature. There is a "glass" transition in all enzymes studied thus far that occurs about -55 degree C, leading to a marked reduction in the collective motions in the proteins. Below this transition, substrate binding and catalysis are greatly diminished, even in fluid media. The transition will be characterized by low-temperature protein crystallography combined with molecular dynamics simulations. The fourth sub-project attempts to apply protein crystallographic methods, including those developed in sub-project three, to unravel the protein-protein interactions and conformational changes in bacterial chemotaxis. The chemotactic protein that is the focus of much of this study, CheY, is a member of a family of regulatory proteins that are phosphorylated on specific aspartic acid residues by protein histidine kinases. Other members of this family include the regulator for alginate production by Pseudomonas in cystic fibrosis. The remaining four projects aim to determine the structures and mechanism of four enzymes, including alanine racemase.